PROS Y Martono, Sugeng R, Abdul R, S Martono Optimum ultrasound fulltext
Proceedings of the IConSSE FSM SWCU (2015), pp. BC.149–155
BC.149
ISBN: 978-602-1047-21-7
Optimum ultrasound extraction of stevioside and rebaudioside a from
Stevia rebaudiana leaves on isocratic RP-HPLC analysis
Yohanes Martonoa,c*, Sugeng Riyantob, Abdul Rohmanb, Sudibyo Martonob
Doctorate Student of Faculty of Pharmacy, Gadjah Mada University,
Sekip Utara, Yogyakarta 55221, Indonesia
b
Faculty of Pharmacy, Gadjah Mada University, Sekip Utara, Yogyakarta 55221, Indonesia
c
Chemistry Department, Satya Wacana Christian University,
Jl. Diponegoro No. 52 60, Salatiga 50733, Indonesia
a
Abstract
The important factor on HPLC analysis is sample preparation. Optimum sample extraction
yield accurate quantitation of respected compounds. This study evaluated the application
of ultrasound extraction method for solid-liquid extraction of stevioside and rebaudioside
A from Stevia rebaudiana leaves. Different solvents extraction (water, methanol, ethanol
40 70%, v/v in water, and mobile phase), times (5, 15, 30, and 60 minutes), and frequent
of re-extraction (2 times) were used. The extraction was done on 40 C. Stevioside and
rebaudioside A content were determined using isocratic RP-HPLC. The result showed that
the optimum condition for stevioside and rebaudioside A extraction was using ethanol
60% (v/v in water) for 15 minutes in twice re-extraction. There were optimal time and reextraction frequent in ultrasound extraction to yield maximum respected compounds on
isocratic RP-HPLC sample preparation.
Keywords stevioside, rebaudioside A, Stevia rebaudiana, extraction, RP-HPLC
1.
Introduction
Stevioside and rebaudioside A were dominant steviol glycoside in Stevia rebaudiana
leaves. These compounds were widespread used as natural low calorie sweetener in various
food and beverages product (Liu et al., 2010). Even, stevioside was the potential drug
candidate for diabetes type 2 (Gregersen et al., 2004; Kujur et al., 2010). Purified extract or
isolate of stevioside and rebaudioside A has marketing value if contained more than 80% and
90% of stevioside and rebaudioside A respectively (Gardana et al., 2010). Due to marketing
value, Stevia rebaudiana leaves were needed to be guaranteed for quality in stevioside and
rebaudioside A content. High quality leaves will yield high marketing value of stevioside and
rebaudioside A purified extract. Stevioside and rebaudioside A content in Stevia rebaudiana
leaves were 6 10% and 2 4% respectively (Londhe & Nanaware, 2013) .
HPLC method was developed to determine stevioside and rebaudioside A content in
Stevia rebaudiana leaves (Bergs et al., 2012; Cacciola et al., 2011; Catharino & Santos, 2012;
Dacome et al., 2005; Gardana et al., 2010; Jaworska et al., 2012; Kolb et al., 2001; Minne et
al., 2004; Pól et al., 2007; Shafii et al., 2012; Well et al., 2013; Yang & Chen, 2009; Zhao et al.,
2013). One point different among the developed methods was sample preparation. (Kolb et
*
Corresponding author. Tel.: +628562715085; E-mail address: yohanes.martono@staff.uksw.edu.
SWUP
Optimum ultrasound extraction of stevioside and rebaudioside a from Stevia rebaudiana leaves
on isocratic RP-HPLC analysis
BC.150
al., 2001) used ethanol 70% (v/v in water) for solid-liquid extraction at 70 C. (Dacome et al.,
2005) used boiling water in sample preparation to extract respected compounds. (Yang &
Chen, 2009) and (Shafii et al., 2012) used ethanol 50% and 60% respectively to yield
maximum respected compounds content in sample preparation. Different with others,
(Jaworska et al., 2012) used acetonitrile : water (80 : 20, v/v) for extracting respected
compounds from Stevia rebaudiana leaves. The different conditions of sample preparation
method showed that it needed the optimization on sample preparation method. Perische et
al.(2015) and Liu et al. (2010) reported that ultrasound extraction could extract steviol
glycoside higher than conventional extraction.
Due to sample preparation is one important factor that can influence the accurate
content determination of respected compounds on HPLC analysis, it needed to optimize the
condition of sample preparation. This study evaluated solvent extraction, time, and frequent
of extraction in ultrasound extraction for extracting stevioside and rebaudioside A from
Stevia rebaudiana leaves.
2.
Materials and methods
2.1 Sample and chemicals
Stevia rebaudiana Bert. leaves were obtained from Tawangmangu, Karanganyar,
Central Java, Indonesia. Stevioside and rebaudioside A standard were purchased from WAKO,
Japan with purity > 99.0%. Methanol (HPLC grade), ethanol (Pro-Analysis), Trifluoro acetic
acid (TFA, pro-analysis) and acetonitrile (HPLC grade) were purchased from Merck.
2.2 Instruments
Stevioside and rebaudioside A content was determined using Reverse Phase-HPLC.
Stationary phase was using Eurosphere (250 × 4.6 mm, 5 µm) column with pre-column. HPLC
analysis was performed by Knauer GmBH-Germany Smart Line Series HPLC with Smart Line
UV 2500 A5140 detector, Smart Line Pump 1000 V 7603, sample injector 20 µL Rheodyne
Loop model A1357. Sonicator Krisbow DSA50-GL2-2.5L was used during ultrasound
extraction.
2.3 Chromatographic conditions
Chromatography analysis was performed in isocratic elusion system. Reverse-phase
HPLC analysis was done using Eurosphere (250 mm × 4.6 mm i.d × 5 µm) column with precolumn. Separation of respected compounds was using methanol : water (90 : 10, v/v, pH =
3.0) : acetonitrile : TFA (65 : 35 : 0.01, v/v/v) as mobile phase. Stevioside and rebaudioside A
separation was accomplished at constant flow of 0.6 mL.min-1. The column was thermostated
at 30 oC. Detection of respected compounds was set at 210 nm and the injection volume was
20 µL. Running times analysis system did not exceed 15 min.
2.4 Quantitation
Individual stock standard solutions were prepared at 500 µg mL-1 by exact weighing
and dillution in mobile phase. Concentration series of respected compounds were made in
range 20 120 µg mL-1 of external standard solutions. Callibration curve was made by plotting
area vs concentration at five level concentrations. The external standard callibration curve
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Y. Martono, S. Riyanto, A. Rohman, S. Martono
BC.151
was used to determine stevioside and rebaudioside A content in Stevia rebaudiana leaves
extract.
2.5 Optimization of ultrasound extraction
Dried Stevia rebaudiana leaves were ground with grinder untill formed fine powder.
Extraction was done by sonicating 0.5 gram fine leaves powder and extracting at 40 oC in
various solvent, times, and twice re-extraction. Optimization was performed by varying some
variable:
1. Polarity solvent extraction was optimzed by varying ethanol concentration from 40,
60, 70% and mobile phase to extract respected compounds. Each solvents was used
as solvent extraction in ultrasound extraction for 1 hour at 40oC. Each extract was
filtered using 0.45 nylon microfilter (Whatman Inc.) before injected to HPLC system.
2. Based on certain solvent extraction from previous optimization, period (times)
extraction was optimzed by sonicating dried fine powder sample in various times, 5,
15, 30, and 60 minutes. Each remaining residue was re-extracted in twice with same
procedure on first extraction before. Each fractions from various times and
consecutive extraction was filtered with 0.45 µm microfilter (Whatman Inc.) before
injected to HPLC system.
3. Suitable solvent extraction was ensured by varying solvent extraction with methanol
50%, boiling water and certain concentration of ethanol. Extraction was done by
sonicating dried powder with each solvents for certain times and re-extraction cycle
at 40oC . After sonication, the filtrate was adjusted in 100 mL volumetric flask with
certain solvent. Each extract was filtered using nylon microfiltered (Whatman Inc.)
before injected to HPLC system.
3.
Results and discussion
3.1 External standard callibration curve
Stevioside and rebaudioside A determination was done by using externall standar curve
callibration method. Developed RP-HPLC method gave linear standard curve with R2 > 0.9994
(see Figure 1). This result showed that standard curve used was in good linearity to determine
respected compounds.
(a)
(b)
Figure 1. Standard curve of: (a) stevioside and (b) rebaudioside A.
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Optimum ultrasound extraction of stevioside and rebaudioside a from Stevia rebaudiana leaves
on isocratic RP-HPLC analysis
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3.2 Optimization of ultrasound extraction
Every solvent has different polarity. The suitability of solvent polarity with respected
compounds to be extracted was important factor in extraction. Beside, time and cycle
extraction were also influence the optimum yield of extraction. Different method of extraction
will give different result in quantity of respected compounds. Perische et al. (2015) showed
that microwave and ultrasound extraction methods gave higher extraction yield than
maceration method in stevioside and rebaudioside A content. Different extraction methods
were also has different optimum time to extract respected compunds. Extraction in over time
of optimum time extraction will not give better result in respected compounds content.
Residue after first extraction was still containing respected compounds. The first extraction
could yield 86% of steviol glycoside compounds and reached maximum extraction in second
extraction as far there was no steviol quantifiable in third extraction (less than Limit of
Quantitation, LOQ) (Jentzer et al., 2015).
Ethanol was more suitable in polaratiy to extract steviol glycoside than methanol and
boiling water. Stevioside and rebaudioside A content were higher in ethanol extraction (see
Figure 2). The result was agreeing with (Shafii et al., 2012) that suitable solvent to extract
stevioside and rebaudioside A was ethanol 60%. The result was different from (Dacome et al.,
2005) which used water in sodium phosphate buffer system (pH 7.00) to extract steviol
glycoside from Stevia rebaudiana leaves. The different method extraction will give different
optimum solvent to extract respected compounds.
Figure 2. Stevioside and Rebaudioside A content from Stevia rebaudiana leaves in
various solvent extraction.
Table 1. Stevioside and rebaudioside A content from Stevia rebaudiana leaves in
various concentration solvent.
Solvent
Ethanol 50%
Ethanol 60%
Ethanol 70%
Mobile phase
Rebaudioside A
(%)
1.53
1.69
1.49
1.50
Stevioside
(%)
10.83
11.56
11.32
9.21
Total dominant steviol glycoside
(%)
12.36
13.25
12.81
10.71
The suitabilty of solvent polarity was studied deeply by varying solvent concentration.
This study showed that ethanol 60% was the suitable solvent to extract stevioside and
rebaudioside A from Stevia rebaudiana leaves (see Table 1). Water content in ethanol will
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Y. Martono, S. Riyanto, A. Rohman, S. Martono
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modify its polarity and increase suitability to extract stevioside and rebaudioside A from Stevia
rebaudiana leaves. Compiring with ethanol 50%, 70% and mobile phase, ethanol 60% was
more suitable in polarity to extract stevioside and rebaudioside A. This result indicated that
there was optimum concentration of solvent in ultrasound extraction.
The other important factor in extraction is time. There is optimum time extraction to
extract respected compounds. Different methods will give different optimum time extraction.
Extraction time will influence the extraction yield because the longer extraction time will make
longer contact time between two phases and the increase yield will be obtained (Jentzer et
al., 2015). Figure 3 illustrated that there was optimum time extraction for stevioside but the
rebaudioside A still remain increase for 1 hour period in ultrasound extraction by using ethanol
60% at 40 oC. The optimum time extraction was 15 min that give optimum yield extraction in
stevioside content. Stevioside was reached optimum extraction because of its polarity suitable
with ethanol 60%. Rebaudioside has three sugas bounded at C-19 while stevioside bound two
sugar at the same atom. It means that rebaudioside A was more polar than stevioside. The
polarity of ethanol 60% was more suitable for stevioside than rebaudioside A.
Figure 3. Optimization of extraction time in 3 cycle for: (a) stevioside and (b)
rebaudioside A.
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Optimum ultrasound extraction of stevioside and rebaudioside a from Stevia rebaudiana leaves
on isocratic RP-HPLC analysis
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Figure 3 also illustrated the influence of extraction cycles. The cycles will influence
diffusion equillibrium of respected compounds. The addition of new solvent in cycles addition
will disturb diffusion equillbrium and make longer time to diffuse the respected compounds
from matrix sample to extraction solvent (Jentzer et al., 2015). Addition cycles untill 3 cycles
can increase extraction yield of stevioside and rebaudioside A content. In stevioside
extraction, the first cycle extraction for 15 min could extract approximately 86% stevioside.
The third cycles of extraction remain only 1.65% for 15 min extraction. In other hand,
effectivenes rebaudioside A extraction was lower than stevioside for 15 min extraction.
Rebaudioside A still increase for 60 min extraction. The first cycle of 60 min extraction could
yield 87.96% and the third cycle remain 1.28% rebaudioside A. The compound s polarity
influenced extractable ability. There for stevioside was faster extracted than rebaudioside A
because its polarity was suitable with ethanol 60%. The longer contact time will increase
disolution of respected compounds from matrix to sample and increase the content of
respected compounds.
The developed sample extraction method was more effective to extract stevioside and
rebaudioside A compounds. The method developed was shorter in ultrasound extraction than
method developed by (Jaworska et al., 2012). The different method was extraction solvent.
Jaworska used acetonitrile : water (30 : 70, v/v) for 1 hour or 60 min to extract steviol glycoside
by ultrasound extraction. It means that suitable polarity will influenced efectiveness of
extraction. The other study showed longer (3 hours) extraction time by water as extraction
solvent (Bergs et al., 2012). The method developed by Bergs et al (2012) used shaker to extract
steviol glycoside. The result showed that ultrasound extraction was more effective to extract
stevioside and rebaudioside A from Stevia rebaudiana leaves. Chromatogram of stevioside
and rebaudioside A from Stevia leaves was shown in Figure 4.
Figure 4. Chromatogram of Stevia levaes on isocratic RP-HPLC analysis with ultrasound
extraction showed rebaudioside A (1) and stevioside (2) as respected compounds.
4.
Conclusion
Sample extraction for stevioside and rebaudioside A analysis by RP-HPLC were
influenced by extraction method, extraction solvent polarity, extraction time and number of
cycle extraction. The optimum condition of extraction were using ultrasound extraction
method with ethanol 60% as extraction solvent for 15 min in 3 cycles extraction at 40 oC.
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References
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Bergs, D., Burghoff, B., Joehnck, M., Martin, G., & Schembecker, G. (2012). Fast and isocratic HPLCmethod for steviol glycosides analysis from Stevia rebaudiana leaves. J. Für Verbraucherschutz
Leb., 7, 147 154.
Cacciola, F., Delmonte, P., Jaworska, K., Dugo, P., Mondello, L., & Rader, J.I. (2011). Employing ultra
high pressure liquid chromatography as the second dimension in a comprehensive twodimensional system for analysis of Stevia rebaudiana extracts. J. Chromatogr. A, 1218, 2012
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Catharino, R.R., & Santos, L.S. (2012). On-line monitoring of stevioside sweetener hydrolysis to steviol
in acidic aqueous solutions. Food Chem., 133, 1632 1635.
Dacome, A.S., Da Silva, C.C., Da Costa, C.E., Fontana, J.D., Adelmann, J., & Da Costa, S.C., (2005). Sweet
diterpenic glycosides balance of a new cultivar of Stevia rebaudiana (Bert.) Bertoni: Isolation and
quantitative distribution by chromatographic, spectroscopic, and electrophoretic methods.
Process Biochem., 40, 3587 3594.
Gardana, C., Scaglianti, M., & Simonetti, P., (2010). Evaluation of steviol and its glycosides in Stevia
rebaudiana leaves and commercial sweetener by ultra-high-performance liquid
chromatography-mass spectrometry. J. Chromatogr. A, 1217, 1463 1470.
Gregersen, S., Jeppesen, P.B., Holst, J.J., & Hermansen, K., (2004). Antihyperglycemic effects of
stevioside in type 2 diabetic subjects. Metabolism, 53, 73 76.
Jaworska, K., Krynitsky, A.J., Rader, J.I., (2012). Simultaneous analysis of steviol and steviol glycosides
by liquid chromatography with ultraviolet detection on a mixed-mode column: application to
Stevia plant material and Stevia-containing dietary supplements. J. AOAC Int., 95, 1588 1596.
Jentzer, J.-B., Alignan, M., Vaca-Garcia, C., Rigal, L., Vilarem, G., (2015). Response surface methodology
to optimise Accelerated Solvent Extraction of steviol glycosides from Stevia rebaudiana Bertoni
leaves. Food Chem., 166, 561 567. doi:10.1016/j.foodchem.2014.06.078.
Kolb, N., Herrera, J.L., Ferreyra, D.J., Uliana, R.F., (2001). Analysis of sweet diterpene glycosides from
Stevia rebaudiana: Improved HPLC method. J. Agric. Food Chem., 49, 4538 4541.
Kujur, R.S., Singh, V., Ram, M., Yadava, H.K., Singh, K.K., Kumari, S., and et al., (2010). Antidiabetic
Activity and Phytochemical Screening of Crude Extract of Stevia Rebaudiana in Alloxan-induced
Diabetiis Rats. Pharmacogn. J., 2, 27 32.
Liu, J., Li, J., Tang, J., (2010). Ultrasonically assisted extraction of total carbohydrates from Stevia
rebaudiana Bertoni and identification of extracts. Food Bioprod. Process., 88, 215 221.
Londhe, S.V., Nanaware, S.M., (2013). HPTLC Method for Simultaneous Analysis of Stevioside and
Rebaudioside-A in Stevia rebaudiana. J. AOAC Int., 96, 24 26.
Minne, V.J., Compernolle, F., Toppet, S., Geuns, J.M., (2004). Steviol quantification at the picomole
level by high-performance liquid chromatography. J. Agric. Food Chem., 52, 2445 2449.
Periche, A., Castelló, M.L., Heredia, A & Isabel Escriche. (2015). Influence of Extraction Methods on the
Yield of Steviol Glycosides and Antioxidants in Stevia rebaudiana Extracts. Plant Foods Hum Nutr,
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Pól, J., Hohnová, B., Hyötyläinen, T., (2007). Characterisation of Stevia rebaudiana by comprehensive
two-dimensional liquid chromatography time-of-flight mass spectrometry. J. Chromatogr. A,
1150, 85 92.
Shafii, B., Vismeh, R., Beaudry, R., Warner, R., Jones, A.D., (2012). Large-scale profiling of diterpenoid
glycosides from Stevia rebaudiana using ultrahigh performance liquid chromatography/tandem
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Yang, D., Chen, B., (2009). Simultaneous determination of nonnutritive sweeteners in foods by
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SWUP
BC.149
ISBN: 978-602-1047-21-7
Optimum ultrasound extraction of stevioside and rebaudioside a from
Stevia rebaudiana leaves on isocratic RP-HPLC analysis
Yohanes Martonoa,c*, Sugeng Riyantob, Abdul Rohmanb, Sudibyo Martonob
Doctorate Student of Faculty of Pharmacy, Gadjah Mada University,
Sekip Utara, Yogyakarta 55221, Indonesia
b
Faculty of Pharmacy, Gadjah Mada University, Sekip Utara, Yogyakarta 55221, Indonesia
c
Chemistry Department, Satya Wacana Christian University,
Jl. Diponegoro No. 52 60, Salatiga 50733, Indonesia
a
Abstract
The important factor on HPLC analysis is sample preparation. Optimum sample extraction
yield accurate quantitation of respected compounds. This study evaluated the application
of ultrasound extraction method for solid-liquid extraction of stevioside and rebaudioside
A from Stevia rebaudiana leaves. Different solvents extraction (water, methanol, ethanol
40 70%, v/v in water, and mobile phase), times (5, 15, 30, and 60 minutes), and frequent
of re-extraction (2 times) were used. The extraction was done on 40 C. Stevioside and
rebaudioside A content were determined using isocratic RP-HPLC. The result showed that
the optimum condition for stevioside and rebaudioside A extraction was using ethanol
60% (v/v in water) for 15 minutes in twice re-extraction. There were optimal time and reextraction frequent in ultrasound extraction to yield maximum respected compounds on
isocratic RP-HPLC sample preparation.
Keywords stevioside, rebaudioside A, Stevia rebaudiana, extraction, RP-HPLC
1.
Introduction
Stevioside and rebaudioside A were dominant steviol glycoside in Stevia rebaudiana
leaves. These compounds were widespread used as natural low calorie sweetener in various
food and beverages product (Liu et al., 2010). Even, stevioside was the potential drug
candidate for diabetes type 2 (Gregersen et al., 2004; Kujur et al., 2010). Purified extract or
isolate of stevioside and rebaudioside A has marketing value if contained more than 80% and
90% of stevioside and rebaudioside A respectively (Gardana et al., 2010). Due to marketing
value, Stevia rebaudiana leaves were needed to be guaranteed for quality in stevioside and
rebaudioside A content. High quality leaves will yield high marketing value of stevioside and
rebaudioside A purified extract. Stevioside and rebaudioside A content in Stevia rebaudiana
leaves were 6 10% and 2 4% respectively (Londhe & Nanaware, 2013) .
HPLC method was developed to determine stevioside and rebaudioside A content in
Stevia rebaudiana leaves (Bergs et al., 2012; Cacciola et al., 2011; Catharino & Santos, 2012;
Dacome et al., 2005; Gardana et al., 2010; Jaworska et al., 2012; Kolb et al., 2001; Minne et
al., 2004; Pól et al., 2007; Shafii et al., 2012; Well et al., 2013; Yang & Chen, 2009; Zhao et al.,
2013). One point different among the developed methods was sample preparation. (Kolb et
*
Corresponding author. Tel.: +628562715085; E-mail address: yohanes.martono@staff.uksw.edu.
SWUP
Optimum ultrasound extraction of stevioside and rebaudioside a from Stevia rebaudiana leaves
on isocratic RP-HPLC analysis
BC.150
al., 2001) used ethanol 70% (v/v in water) for solid-liquid extraction at 70 C. (Dacome et al.,
2005) used boiling water in sample preparation to extract respected compounds. (Yang &
Chen, 2009) and (Shafii et al., 2012) used ethanol 50% and 60% respectively to yield
maximum respected compounds content in sample preparation. Different with others,
(Jaworska et al., 2012) used acetonitrile : water (80 : 20, v/v) for extracting respected
compounds from Stevia rebaudiana leaves. The different conditions of sample preparation
method showed that it needed the optimization on sample preparation method. Perische et
al.(2015) and Liu et al. (2010) reported that ultrasound extraction could extract steviol
glycoside higher than conventional extraction.
Due to sample preparation is one important factor that can influence the accurate
content determination of respected compounds on HPLC analysis, it needed to optimize the
condition of sample preparation. This study evaluated solvent extraction, time, and frequent
of extraction in ultrasound extraction for extracting stevioside and rebaudioside A from
Stevia rebaudiana leaves.
2.
Materials and methods
2.1 Sample and chemicals
Stevia rebaudiana Bert. leaves were obtained from Tawangmangu, Karanganyar,
Central Java, Indonesia. Stevioside and rebaudioside A standard were purchased from WAKO,
Japan with purity > 99.0%. Methanol (HPLC grade), ethanol (Pro-Analysis), Trifluoro acetic
acid (TFA, pro-analysis) and acetonitrile (HPLC grade) were purchased from Merck.
2.2 Instruments
Stevioside and rebaudioside A content was determined using Reverse Phase-HPLC.
Stationary phase was using Eurosphere (250 × 4.6 mm, 5 µm) column with pre-column. HPLC
analysis was performed by Knauer GmBH-Germany Smart Line Series HPLC with Smart Line
UV 2500 A5140 detector, Smart Line Pump 1000 V 7603, sample injector 20 µL Rheodyne
Loop model A1357. Sonicator Krisbow DSA50-GL2-2.5L was used during ultrasound
extraction.
2.3 Chromatographic conditions
Chromatography analysis was performed in isocratic elusion system. Reverse-phase
HPLC analysis was done using Eurosphere (250 mm × 4.6 mm i.d × 5 µm) column with precolumn. Separation of respected compounds was using methanol : water (90 : 10, v/v, pH =
3.0) : acetonitrile : TFA (65 : 35 : 0.01, v/v/v) as mobile phase. Stevioside and rebaudioside A
separation was accomplished at constant flow of 0.6 mL.min-1. The column was thermostated
at 30 oC. Detection of respected compounds was set at 210 nm and the injection volume was
20 µL. Running times analysis system did not exceed 15 min.
2.4 Quantitation
Individual stock standard solutions were prepared at 500 µg mL-1 by exact weighing
and dillution in mobile phase. Concentration series of respected compounds were made in
range 20 120 µg mL-1 of external standard solutions. Callibration curve was made by plotting
area vs concentration at five level concentrations. The external standard callibration curve
SWUP
Y. Martono, S. Riyanto, A. Rohman, S. Martono
BC.151
was used to determine stevioside and rebaudioside A content in Stevia rebaudiana leaves
extract.
2.5 Optimization of ultrasound extraction
Dried Stevia rebaudiana leaves were ground with grinder untill formed fine powder.
Extraction was done by sonicating 0.5 gram fine leaves powder and extracting at 40 oC in
various solvent, times, and twice re-extraction. Optimization was performed by varying some
variable:
1. Polarity solvent extraction was optimzed by varying ethanol concentration from 40,
60, 70% and mobile phase to extract respected compounds. Each solvents was used
as solvent extraction in ultrasound extraction for 1 hour at 40oC. Each extract was
filtered using 0.45 nylon microfilter (Whatman Inc.) before injected to HPLC system.
2. Based on certain solvent extraction from previous optimization, period (times)
extraction was optimzed by sonicating dried fine powder sample in various times, 5,
15, 30, and 60 minutes. Each remaining residue was re-extracted in twice with same
procedure on first extraction before. Each fractions from various times and
consecutive extraction was filtered with 0.45 µm microfilter (Whatman Inc.) before
injected to HPLC system.
3. Suitable solvent extraction was ensured by varying solvent extraction with methanol
50%, boiling water and certain concentration of ethanol. Extraction was done by
sonicating dried powder with each solvents for certain times and re-extraction cycle
at 40oC . After sonication, the filtrate was adjusted in 100 mL volumetric flask with
certain solvent. Each extract was filtered using nylon microfiltered (Whatman Inc.)
before injected to HPLC system.
3.
Results and discussion
3.1 External standard callibration curve
Stevioside and rebaudioside A determination was done by using externall standar curve
callibration method. Developed RP-HPLC method gave linear standard curve with R2 > 0.9994
(see Figure 1). This result showed that standard curve used was in good linearity to determine
respected compounds.
(a)
(b)
Figure 1. Standard curve of: (a) stevioside and (b) rebaudioside A.
SWUP
Optimum ultrasound extraction of stevioside and rebaudioside a from Stevia rebaudiana leaves
on isocratic RP-HPLC analysis
BC.152
3.2 Optimization of ultrasound extraction
Every solvent has different polarity. The suitability of solvent polarity with respected
compounds to be extracted was important factor in extraction. Beside, time and cycle
extraction were also influence the optimum yield of extraction. Different method of extraction
will give different result in quantity of respected compounds. Perische et al. (2015) showed
that microwave and ultrasound extraction methods gave higher extraction yield than
maceration method in stevioside and rebaudioside A content. Different extraction methods
were also has different optimum time to extract respected compunds. Extraction in over time
of optimum time extraction will not give better result in respected compounds content.
Residue after first extraction was still containing respected compounds. The first extraction
could yield 86% of steviol glycoside compounds and reached maximum extraction in second
extraction as far there was no steviol quantifiable in third extraction (less than Limit of
Quantitation, LOQ) (Jentzer et al., 2015).
Ethanol was more suitable in polaratiy to extract steviol glycoside than methanol and
boiling water. Stevioside and rebaudioside A content were higher in ethanol extraction (see
Figure 2). The result was agreeing with (Shafii et al., 2012) that suitable solvent to extract
stevioside and rebaudioside A was ethanol 60%. The result was different from (Dacome et al.,
2005) which used water in sodium phosphate buffer system (pH 7.00) to extract steviol
glycoside from Stevia rebaudiana leaves. The different method extraction will give different
optimum solvent to extract respected compounds.
Figure 2. Stevioside and Rebaudioside A content from Stevia rebaudiana leaves in
various solvent extraction.
Table 1. Stevioside and rebaudioside A content from Stevia rebaudiana leaves in
various concentration solvent.
Solvent
Ethanol 50%
Ethanol 60%
Ethanol 70%
Mobile phase
Rebaudioside A
(%)
1.53
1.69
1.49
1.50
Stevioside
(%)
10.83
11.56
11.32
9.21
Total dominant steviol glycoside
(%)
12.36
13.25
12.81
10.71
The suitabilty of solvent polarity was studied deeply by varying solvent concentration.
This study showed that ethanol 60% was the suitable solvent to extract stevioside and
rebaudioside A from Stevia rebaudiana leaves (see Table 1). Water content in ethanol will
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Y. Martono, S. Riyanto, A. Rohman, S. Martono
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modify its polarity and increase suitability to extract stevioside and rebaudioside A from Stevia
rebaudiana leaves. Compiring with ethanol 50%, 70% and mobile phase, ethanol 60% was
more suitable in polarity to extract stevioside and rebaudioside A. This result indicated that
there was optimum concentration of solvent in ultrasound extraction.
The other important factor in extraction is time. There is optimum time extraction to
extract respected compounds. Different methods will give different optimum time extraction.
Extraction time will influence the extraction yield because the longer extraction time will make
longer contact time between two phases and the increase yield will be obtained (Jentzer et
al., 2015). Figure 3 illustrated that there was optimum time extraction for stevioside but the
rebaudioside A still remain increase for 1 hour period in ultrasound extraction by using ethanol
60% at 40 oC. The optimum time extraction was 15 min that give optimum yield extraction in
stevioside content. Stevioside was reached optimum extraction because of its polarity suitable
with ethanol 60%. Rebaudioside has three sugas bounded at C-19 while stevioside bound two
sugar at the same atom. It means that rebaudioside A was more polar than stevioside. The
polarity of ethanol 60% was more suitable for stevioside than rebaudioside A.
Figure 3. Optimization of extraction time in 3 cycle for: (a) stevioside and (b)
rebaudioside A.
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Optimum ultrasound extraction of stevioside and rebaudioside a from Stevia rebaudiana leaves
on isocratic RP-HPLC analysis
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Figure 3 also illustrated the influence of extraction cycles. The cycles will influence
diffusion equillibrium of respected compounds. The addition of new solvent in cycles addition
will disturb diffusion equillbrium and make longer time to diffuse the respected compounds
from matrix sample to extraction solvent (Jentzer et al., 2015). Addition cycles untill 3 cycles
can increase extraction yield of stevioside and rebaudioside A content. In stevioside
extraction, the first cycle extraction for 15 min could extract approximately 86% stevioside.
The third cycles of extraction remain only 1.65% for 15 min extraction. In other hand,
effectivenes rebaudioside A extraction was lower than stevioside for 15 min extraction.
Rebaudioside A still increase for 60 min extraction. The first cycle of 60 min extraction could
yield 87.96% and the third cycle remain 1.28% rebaudioside A. The compound s polarity
influenced extractable ability. There for stevioside was faster extracted than rebaudioside A
because its polarity was suitable with ethanol 60%. The longer contact time will increase
disolution of respected compounds from matrix to sample and increase the content of
respected compounds.
The developed sample extraction method was more effective to extract stevioside and
rebaudioside A compounds. The method developed was shorter in ultrasound extraction than
method developed by (Jaworska et al., 2012). The different method was extraction solvent.
Jaworska used acetonitrile : water (30 : 70, v/v) for 1 hour or 60 min to extract steviol glycoside
by ultrasound extraction. It means that suitable polarity will influenced efectiveness of
extraction. The other study showed longer (3 hours) extraction time by water as extraction
solvent (Bergs et al., 2012). The method developed by Bergs et al (2012) used shaker to extract
steviol glycoside. The result showed that ultrasound extraction was more effective to extract
stevioside and rebaudioside A from Stevia rebaudiana leaves. Chromatogram of stevioside
and rebaudioside A from Stevia leaves was shown in Figure 4.
Figure 4. Chromatogram of Stevia levaes on isocratic RP-HPLC analysis with ultrasound
extraction showed rebaudioside A (1) and stevioside (2) as respected compounds.
4.
Conclusion
Sample extraction for stevioside and rebaudioside A analysis by RP-HPLC were
influenced by extraction method, extraction solvent polarity, extraction time and number of
cycle extraction. The optimum condition of extraction were using ultrasound extraction
method with ethanol 60% as extraction solvent for 15 min in 3 cycles extraction at 40 oC.
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